Gli autori ringraziano Vasia VAGIN per l&#39;introduzione di questa tecnica nel nostro laboratorio.

<ol><li> Fare un vetrino da microscopio in vetro con uno sfondo nero: Tagliare carta da 20 # bianco delle stesse dimensioni del vetrino da microscopio. Colore un lato della slide-nero Taglia carta con un pennarello indelebile (ad esempio, Sharpie). Applicare la colla bianca (Elmer) per il lato nero e aderire la carta alla diapositiva. La presentazione può essere riutilizzato. Utilizzando ¾ &quot;tape label (Fisherbrand), fissare il vetrino da microscopio, vetro laterale alto, al pad CO 2 volare a fissarlo. </li><li> Raccogliere 0-3 vola maschio giorno. </li><li> Preparare un secchio di ghiaccio, PBS (PBS), aghi di dissezione. </li><li> Anestetizzare 50-100 vola utilizzando un flusso di CO 2 e trasferirli dalla bottiglia o flacone per il pad volare vicino al vetrino da microscopio. </li><li> Pipettare ~ 50 microlitri ghiaccio PBS freddo in una goccia nel mezzo del vetrino da microscopio. </li><li> Delicatamente prendere un moscerino maschio con la dissezione ago-dip l&#39;ago dissezione in PBS, tocco gentile lato ventrale (cioè, in prossimità delle ali) della mosca con l&#39;ago posto in una goccia di PBS. </li><li> Posto un ago al centro della mosca, tra il torace e l&#39;addome, spingendo con decisione. Posizionare l&#39;ago altro tra il sesto (A6) e settimo (A7) segmenti addominali (le strisce secondo e il terzo dal basso); tirare l&#39;addome di distanza dal torace. </li><li> Stuzzicare i testicoli a parte altri organi, in particolare dell&#39;intestino. Soprattutto in mutanti bianco, testicoli e intestino simile. Differenze principali sono che testicoli vengono sempre a coppie, ognuna testicolo è un percorso a spirale ed è più di colore bianco che nell&#39;intestino. </li><li> Conservare i testicoli nella gelida PBS in una provetta polipropilene. </li></ol> Rappresentante dei risultati: <img src="/files/ftp_upload/2641/2641fig1.jpg" alt="Figura 1" /> Figura 1. Vista dorsale del sistema riproduttivo maschile, da flybase.org/reports/FBim0000074.html. Flybase attribuzione:... &quot;Dopo Figura 38 A a pagina 508 di Miller (1950) A. Miller, L&#39;anatomia interna e istologia della imago di Drosophila melanogaster In M. Demerec, editore, Biologia della Drosophila, capitolo 6, pagine 420 - 534. Cold Spring Harbor Laboratory Press, edizione facsimile, 1994. Copyright 1994 da Cold Spring Harbor Laboratory Press. Utilizzato per gentile concessione del Cold Spring Harbor Laboratory (CSHL) Premere. Vedere <a href="http://www.cshlpress.com">http://www.cshlpress.com</a> &quot;.

<ol>
	<li>Singh, S. R., Hou, S. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistological+Techniques+for+Studying+the+Drosophila+Male+Germline+Stem+Cell.">Immunohistological Techniques for Studying the Drosophila Male Germline Stem Cell.</a> Methods Mol Biol. 450, 45-59 (2008).</li></ol>

Nessun conflitto di interessi dichiarati.

Forniamo un protocollo per isolare i testicoli di adulto maschio di Drosophila melanogaster. Perché la tecnica richiede coordinamento proprio due mani, l&#39;abilità viene solo con la pratica.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>Dissecting needle</td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>S17346</td>
<td>&nbsp;</td>
<tr>
<td>Micro Slides, Plain </td>
<td>Tool</td>
<td>Fisher Scientific</td>
<td>12-553</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Light</td>
<td>Tool</td>
<td>Schott-Fostec</td>
<td>KO-1500</td>
<td>&nbsp;</td>
<tr>
<td>CO2 pad</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>59-172</td>
<td>&nbsp;</td>
<tr>
<td>Benchtop Flowbuddy Complete System</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>59-122WCU</td>
<td>&nbsp;</td>
<tr>
<td>Bubble Humidifier</td>
<td>Tool</td>
<td>Salter Labs</td>
<td>7100</td>
<td>&nbsp;</td>
<tr>
<td>Ball filler needle</td>
<td>Tool</td>
<td>Genesee Scientific</td>
<td>54-119</td>
<td>&nbsp;</td>
<tr>
<td>&frac34;&#x201D; Label Tape</td>
<td>Supply</td>
<td>Fisher Scientific</td>
<td>15-901-5B</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

isolation of drosophila melanogaster testes

I testicoli di Drosophila melanogaster fornire un importante modello per lo studio della manutenzione e la differenziazione delle cellule staminali, la meiosi, e soma-germinale interazioni. I testicoli sono tipicamente isolate da maschi adulti 0-3 giorni dopo eclosion dal caso pupa. I testicoli di wild-type mosche sono facilmente distinguibili da altri tessuti perché sono gialle, ma i testicoli del bianco mosche mutanti, un background genetico comune per esperimenti di laboratorio sono simili in forma e colore per l&#39;intestino volare. Esecuzione di dissezione su un vetrino di vetro con uno sfondo nero rende l&#39;identificazione dei testicoli molto più semplice. Testicoli vengono rimossi dalle mosche utilizzando aghi dissezione. Rispetto ai protocolli che utilizzano pinze per la dissezione dei testicoli, il nostro metodo è molto più veloce, che consente una ben praticata individuale di sezionare testicoli 200-300 wild-type vola per ora, cedendo 400-600 testicoli. Testicoli dalle mosche bianche o da mutanti che riducono le dimensioni dei testicoli sono più difficili da analizzare e in genere resa testicoli 200-400 all&#39;ora.

Watch this Scientific Journal Video about Isolation of Drosophila melanogaster Testes at JoVE.com

Isolation of Drosophila melanogaster Testes

Drosophila melanogaster testicoli possono essere rapidamente ed efficacemente isolato da maschi adulti con aghi dissezione. Con la pratica, si può facilmente isolare in uno o due giorni una quantità di testicoli sufficiente per l&#39;analisi del DNA o RNA mediante sequenziamento throughput elevato o più tradizionali metodi di biologia molecolare o di proteine ​​per i test degli anticorpi-o a base di enzimi.

Isolamento di Drosophila melanogaster Testicoli

The testes of Drosophila melanogaster provide an important model for the study of stem cell maintenance and differentiation, meiosis, and soma-germline ...

isolation-of-drosophila-melanogaster-testes

Research

JoVE Journal

Biology

Isolation of Drosophila melanogaster Testes

32.3K Views.  University of Massachusetts Medical School. Drosophila melanogaster testicoli possono essere rapidamente ed efficacemente isolato da maschi adulti con aghi dissezione. Con la pratica, si può facilmente isolare in uno o due giorni una quantità di testicoli sufficiente per l'analisi del DNA o RNA mediante sequenziamento throughput elevato o più tradizionali metodi di biologia molecolare o di proteine ​​per i test degli anticorpi-o a base di enzimi.

Video: Isolation of Drosophila melanogaster Testes

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

Dissezione del sistema nervoso centrale larvali in Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Ricerca

Biologia

High-Resolution Video Tracking of Locomotion in Adult Drosophila Melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field. Studying locomotor behavior in Drosophila melanogaster relies on the ability to be able to quantify changes in motion during or in response to a given task. For this reason, a high-resolution video tracking system, such as the one we describe in this paper, is a valuable tool for measuring locomotion in real-time. Our protocol involves the use of an initial air pulse to break the flies momentum, followed by a thirty second filming period in a square chamber. A tracking program is then used to calculate the instantaneous speed of each fly within the chamber in 10 msec increments. Analysis software then compiles this data, and outputs a variety of parameters such as average speed, max speed, time spent in motion, acceleration, etc. This protocol will discuss proper feeding and management of flies for behavioral tasks, handling flies without anesthetization or immobilization, setting up a controlled environment, and running the assay from start to finish.

The study of complex locomotor behavior in Drosophila melanogaster is dependent upon the ability to quantify changes in a given fly's movement. This article demonstrates how to do this using a high-resolution tracking system.

Monitoraggio ad alta risoluzione video di locomozione per adulti in Drosophila melanogaster

Flies provide an important model for studying complex behavior due to the plethora of genetic tools available to researchers in this field.  Studying ...

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained and restrained whole larvae. For direct control of the environment around the heart another approach utilizes the dissected larvae and removal of the internal organs in order to bathe the heart in desired compounds. The exposed heart also allows membrane potentials to be monitored which can give insight of the ionic currents generated by the myocytes and for electrical conduction along the heart tube. These approaches have various advantages and disadvantages for future experiments that are discussed. The larval heart preparation provides an additional model besides the Drosophila skeletal NMJ to investigate the role of intracellular calcium regulation on cellular function. Learning more about the underlying ionic currents that shape the action potentials in myocytes in various species, one can hope to get a handle on the known ionic dysfunctions associated to specific genes responsible for various diseases in mammals.

Monitoring Heart Function in Larval Drosophila melanogaster for Physiological Studies

We present various ways to monitor heart function in the larva of Drosophila for assessing questions dealing with the function of gap junctions, ion channel mutations, modulation of pacemaker activity and pharmacological studies.

Il monitoraggio delle funzioni cardiaca in larvali Drosophila melanogaster Per gli Studi fisiologici

We present various methods to record cardiac function in the larval Drosophila. The approaches allow heart rate to be measured in unrestrained  and  ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

Immagini dal vivo di Drosophila melanogaster Migrazioni Hemocyte embrionali

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Dissezione di Oenocytes da adulti Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Measurement of Lifespan in Drosophila melanogaster

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced physical performance and increased risk of disease. Individual aging is manifest at the population level as an increase in age-dependent mortality, which is often measured in the laboratory by observing lifespan in large cohorts of age-matched individuals. Experiments that seek to quantify the extent to which genetic or environmental manipulations impact lifespan in simple model organisms have been remarkably successful for understanding the aspects of aging that are conserved across taxa and for inspiring new strategies for extending lifespan and preventing age-associated disease in mammals.
The vinegar fly, Drosophila melanogaster, is an attractive model organism for studying the mechanisms of aging due to its relatively short lifespan, convenient husbandry, and facile genetics. However, demographic measures of aging, including age-specific survival and mortality, are extraordinarily susceptible to even minor variations in experimental design and environment, and the maintenance of strict laboratory practices for the duration of aging experiments is required. These considerations, together with the need to practice careful control of genetic background, are essential for generating robust measurements. Indeed, there are many notable controversies surrounding inference from longevity experiments in yeast, worms, flies and mice that have been traced to environmental or genetic artifacts1-4. In this protocol, we describe a set of procedures that have been optimized over many years of measuring longevity in Drosophila using laboratory vials. We also describe the use of the dLife software, which was developed by our laboratory and is available for download (<a href="http://sitemaker.umich.edu/pletcherlab/software">http://sitemaker.umich.edu/pletcherlab/software</a>). dLife accelerates throughput and promotes good practices by incorporating optimal experimental design, simplifying fly handling and data collection, and standardizing data analysis. We will also discuss the many potential pitfalls in the design, collection, and interpretation of lifespan data, and we provide steps to avoid these dangers.

Measurement of Lifespan in Drosophila melanogaster

Drosophila melanogaster is a powerful model organism for exploring the molecular basis of longevity regulation. This protocol will discuss the steps involved in generating a reproducible, population-based measurement of longevity as well as potential pitfalls and how to avoid them.

Misura della durata della vita in<em&gt; Drosophila melanogaster</em

Aging is a phenomenon that results in steady physiological deterioration in nearly all organisms in which it has been examined, leading to reduced ...

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both the female and male germ lines of Drosophila have contributed greatly to the current understanding of meiosis as well as stem cell biology. Excellent protocols are available in the literature for the isolation and imaging of Drosophila ovaries&#160;and testes3-12. Herein, methods for the dissection and preparation of Drosophila testes for microscopic analysis are described with an accompanying video demonstration. A protocol for isolating testes from the abdomen of adult males and preparing slides of live tissue for analysis by phase-contrast microscopy as well as a protocol for fixing and immunostaining testes for analysis by fluorescence microscopy are presented. These techniques can be applied in the characterization of Drosophila mutants that exhibit defects in spermatogenesis as well as in the visualization of subcellular localizations of proteins.

Cytological Analysis of Spermatogenesis: Live and Fixed Preparations of Drosophila Testes

Methods for isolating and preparing Drosophila testes samples (live and fixed) for imaging by phase-contrast and fluorescence microscopy are described herein.&#160;

L&#39;analisi citologica della spermatogenesi: Preparati Live e fisse di<em&gt; Drosophila</em&gt; Testicoli

Drosophila melanogaster is a powerful model system that has been widely used to elucidate a variety of biological processes. For example, studies of both ...

Imaging Through the Pupal Case of Drosophila melanogaster

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled analysis of cell and developmental biology from a variety of methodological angles. Live imaging is an emerging method for observing dynamic cell processes, such as cell division or cell motility. Having isolated mutations in uncharacterized putative cell cycle proteins it became essential to observe mitosis in situ using live imaging. Most live imaging studies in Drosophila have focused on the embryonic stages that are accessible to manipulation and observation because of their small size and optical clarity. However, in these stages the cell cycle is unusual in that it lacks one or both of the gap phases. By contrast, cells of the pupal wing of Drosophila have a typical cell cycle and undergo a period of rapid mitosis spanning about 20 hr of pupal development. It is easy to identify and isolate pupae of the appropriate stage to catch mitosis in situ. Mounting intact pupae provided the best combination of tractability and durability during imaging, allowing experiments to run for several hours with minimal impact on cell and animal viability. The method allows observation of features as small as, or smaller than, fly chromosomes. Adjustment of microscope settings and the details of mounting, allowed extension of the preparation to visualize membrane dynamics of adjacent cells and fluorescently labeled proteins such as tubulin. This method works for all tested fluorescent proteins and can capture submicron scale features over a variety of time scales. While limited to the outer 20 &#181;m of the pupa with a conventional confocal microscope, this approach to observing protein and cellular dynamics in pupal tissues in vivo may be generally useful in the study of cell and developmental biology in these tissues.

Imaging Through the Pupal Case of Drosophila melanogaster

This paper demonstrates the use of a fast scanning confocal microscope to image cell behavior directly through the puparium. By leaving the pupal case intact, this method allows observation and measurement of dynamic cell processes at a stage of Drosophila development that is difficult to study directly.

Imaging attraverso il caso pupa di<em&gt; Drosophila melanogaster</em

The longstanding use of Drosophila as a model for cell and developmental biology has yielded an array of tools. Together, these techniques have enabled ...

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called imaginal discs. These discs give rise to a high percentage of adult structures that are found within the adult fly. Here we describe a protocol that has been optimized to recover these discs and prepare them for analysis with antibodies, transcriptional reporters and protein traps. This procedure is best suited for thin tissues like imaginal discs, but can be easily modified for use with thicker tissues such as the larval brain and adult ovary. The written protocol and accompanying video will guide the reader/viewer through the dissection of third instar larvae, fixation of tissue, and treatment of imaginal discs with antibodies. The protocol can be used to dissect imaginal discs from younger first and second instar larvae as well. The advantage of this protocol is that it is relatively short and it has been optimized for the high quality preservation of the dissected tissue. Another advantage is that the fixation procedure that is employed works well with the overwhelming number of antibodies that recognize Drosophila proteins. In our experience, there is a very small number of sensitive antibodies that do not work well with this procedure. In these situations, the remedy appears to be to use an alternate fixation cocktail while continuing to follow the guidelines that we have set forth for the dissection steps and antibody incubations.

Dissection and Immunostaining of Imaginal Discs from Drosophila melanogaster

The adult structures of Drosophila are derived from sac-like structures called imaginal discs. Analysis of these discs provides insight into many developmental processes including tissue determination, compartment boundary establishment, cell proliferation, cell fate specification, and planar cell polarity. This protocol is used to prepare imaginal discs for light/fluorescent microscopy.

Dissezione e Immunostaining di immaginale dischi da<em&gt; Drosophila melanogaster</em

A significant portion of post-embryonic development in the fruit fly, Drosophila melanogaster, takes place within a set of sac-like structures called ...

Maintenance of a Drosophila melanogaster Population Cage

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life cycle of Drosophila enables collection of large quantities of material from embryos, larvae, pupae and adult flies, in a synchronized way, at a low economic cost. A major strategy for propagating large numbers of flies is the use of a fly population cage. This useful and common tool in the Drososphila community is an efficient way to regularly produce milligrams to tens of grams of embryos, depending on uniformity of developmental stage desired. While a population cage can be time consuming to set up, maintaining a cage over months takes much less time and enables rapid collection of biological material in a short period. This paper describes a detailed and flexible protocol for the maintenance of a Drosophila melanogaster population cage, starting with 1.5 g of harvested material from the previous cycle.

Maintenance of a Drosophila melanogaster Population Cage

This manuscript reports a detailed protocol for culturing, on a regular basis, a population of Drosophila melanogaster using a fly population cage.

Mantenimento di un<em&gt; Drosophila melanogaster</em&gt; Cage Popolazione

Large quantities of DNA, RNA, proteins and other cellular components are often required for biochemistry and molecular biology experiments. The short life ...